Pharmaceutical compositions and preparations for treatment of metabolic bone disease

ABSTRACT

The present invention relates to a pharmaceutical composition for the treatment of metabolic bone disease and the method of preparation thereof, and more particularly, to an improved pharmaceutical composition for the therapeutic treatment of metabolic disease and the method of preparation thereof, wherein said composition is prepared as a composite pharmaceutical agent which comprises calcitriol; which reduces the rate of spine fractures and increases bone density; alendronate, a bone resorption inhibitor, as two main active ingredients in an optimal mixing ratio to exert the greatest synergistic therapeutic effect; and adequate amount of other additives such as a resorption fortifier of alendronate. Thereof, the pharmaceutical composition according to the present invention can inhibit hypercalcemia caused when administered by calcitriol alone, compensate the inhibitory activity of bone remodeling caused by alendronate due to the presence of calcitriol, and improve drug compliance associated with the usual difficulty in administration as well as a side effect in esophagus, thus effectively preventing the occurence of osteoporosis.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a composite pharmaceutical compositionfor the treatment of metabolic bone disease and the method ofpreparation thereof, wherein said composition is prepared as a compositepharmaceutical agent comprising calcitriol, alendronate, and adequateamount of other additives. The pharmaceutical composition according tothe present invention can inhibit hypercalcemia caused when administeredwith calcitriol alone, compensate the inhibitory activity of boneremodeling caused by alendronate due to the presence of calcitriol, andalso improve drug compliance, thus effectively preventing the occurrenceof osteoporosis.

2. Description of the Related Arts

Osteoporosis is a systemic metabolic bone disease characterized by adecrease in bone mass as well as a microstructural change in bonewithout affecting the chemical composition of bone itself which resultsin increased susceptibility to bone fractures. In particular, senilepatients are known to be more vulnerable to bone fractures, which oftenresult from even minor physical shocks; in fact, approximately 1.3million out of 25 million osteoporosis patients in the U.S. are known tosuffer from bone fractures each year, which accounts for almost 10% oftotal U.S. population.

Osteoporosis can be divided into type I and type II. Type I osteoporosisoccurs most frequently in postmenopausal women and is characterized byhaving a marked increase of bone resorption due to deficiency ofestrogen. It is very marked increase of bone resorption due todeficiency of estrogen. It is very common to observe type I osteoporosispatients lose trabecular bone and this causes frequent fractures ofspines or wrists. Meanwhile, type II osteoporosis usually occurs inelderly people or in males and is characterized by a drastic decrease inbone remodeling capability rather than an increase in bone resorption.However, the increase in bone resorption as well as the decrease incalcium absorption by osteoclasts are known to be common to both typesof osteoporosis.

Therapeutic agents employed in treating osteoporosis in clinical fieldsare generally divided into two groups; a group that can expedite boneremodeling and another that can prevent bone resorption. The examples ofactive therapeutic agents that can promote fast bone remodeling includeanabolic steroids, fluoride, vitamin D and parathyroid hormones. Withthe recent introduction of the mechanism of bone formation, vitamin Dderivatives have been the object of the public attention because oftheir roles in mediating balanced bone mass. Calcitriol, being one ofthe active vitamin D₃ derivatives expressed as 1,25-(OH)₂ D₃, is one ofthe above-mentioned bone remodeling activators, and is also a hormonewhich enables to prevent osteoporosis because it can not only increasecalcium absorption from the intestinal tract but also can inhibit thedecrease of bone mass. However, calcitriol is also shown to causehypercalcemia when used alone. Hypercalcemia, a metabolic diseasecharacterized by having a plasma calcium concentration of 11.0 mg/dL orabove in blood, is one of the most common life-threatening diseaseclosely associated with fatal disease such as a tumor. In fact,calcitriol induces an increase of calcium concentration in blood as wellas the abnormal control of calcium concentration by bone metabolism thusresulting in hypercalcemia. The symptoms of hypercalcemia or calciumaddiction (a severe case of hypercalcemia with prolonged duration) dueto side effects of calcitriol and overdose of vitamin D are very similarto that shown in hypercalcemia. Hypercalcemia occasionally entails acutesymptoms such as anorexia, headache, emesis, constipation, and chronicsymptoms of hypercalcemnia include dystrophy, paresthesia, pyrexia withthirst, hyperurisis, dehydration, acedia, groweighth retardation,urinary infection, etc. Patients with hypercalcemia often experience tohave complications and this adds difficulty in treating disease such asosteoporosis.

The conventional pharmaceutical agents used in treating osteoporosis asbone resorption inhibitors include estrogen, calcitonin, bisphosphonateand ipriflavone.

Japanese Patent Publication No. 7-330613 discloses a bone remodelingactivator, which contains alendronate, a kind of bisphosphonates, as anactive agent and can activate the calcification of osteoblasts as wellas the formation of bone matrix in the presence of dihydroxy vitamin D₃.

French Patent Application No. 884628 discloses a pharmaceutical agentfor oral administration which uses bisphosphonate as a substrate andcontains an adequate amount of sodium lauryl sulfate.

Application Ser. No. 09/125,372 discloses enteric coated tablets thatcontain bisphosphonates.

However, the above-mentioned bone resorption inhibitors have not beensuccessful in sufficiently improving the bone mass of patients withalready progressed osteoporosis and thus there has been along-awaitedneed for the development of very effective bone remodeling activators.

A recent Italian report discloses a method of combined administration ofboth alendronate and calcitriol [Clinical Drug Investigation (1998)March, 15(3), 235-244]. In this report, alendronate and calcitriol werenot administered as a composite tablet but were administeredindependently. For example, alendronate was administered once a daywhile calcitriol was administered twice a day with a certain timelyinterval allowed between each administration.

Furthermore, the alendronate must be administered before meals becausethe pharmaceutical effect was shown much decreased due to poorabsorption when they were taken after meals.

Considering that most osteoporosis patients are elderly people and thatmost of them also take various kinds of drugs daily, it appears veryplausible that the patients often forget to take one or two drugs thatthey are supposed to take because of the rather complicated nature ofadministration of the above drugs and thus the effects of slidadministration also remain questionable.

Alendronate can cause a local stimulation on mucous membranes at theupper gastrointestinal tract and this often results in a few sideeffects in esophagus such as esophagitis, esophageal ulcer, esophagealerosion. Therefore, it becomes necessary for patients to take insufficient amount of water when they take a drug containing alendronateso that the drug they are taking can pass through the esophagus aspromptly as possible and thus keeping esophageal stimulation to theminimal level. And his has been also raised as a burden to most patientsthat lowers drug compliance.

In the Conference of Japanese and American Glaucoma Societies' held inVancouver, Canada in January 1997, it was announced that the final goalof developing composite pharmaceutical agents was to improve drugcompliance. In the ‘Fifth Conference on Retroviruses and opportunisticinfection’ which was held in Chicago on Feb. 4, 1998, it was shown thatthe effect of Combivir, a composite pharmaceutical agent containing bothlamivudine and zidovudine wherein the pharmaceutical mechanisms differfrom each other, had an improved therapeutic effect of 96% as comparedto 72% of effect obtained when lamivudine and zidovudine wereadministered independently as a separate drug, respectively, and herethe improvement in therapeutic effect is speculated to be due to theimprovement in drug compliance of the patient to the compositepharmaceutical agent.

There have been no studies conducted on the therapeutic treatment ofosteoporosis with respect to preparation of composite tablets containingboth calcitriol and alendronate, and instead there have been many linesof studies focused on the effect of alendronate as a therapeutic agenttreating hypercalcemia caused by vitamin D intake. Therefore, it is inhigh demand to develop composite pharmaceutical agents with advancedformulation technologies for the treatment of osteoporosis which can notonly minimize side effects such as hypercalcemia, caused when calcitriolwas administered alone, but can prevent/treat osteoporosis with a littledose and enhance drug compliance, which is usually low in conventionaldrugs, by remedying the rather complicated way of administration andpreventing esophageal side effects.

SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical composition for thetreatment of metabolic disease and the method of preparation thereof,and more particularly, to an improved pharmaceutical composition for thetherapeutic treatment of metabolic disease and the method of preparationthereof, wherein said composition is prepared as a compositepharmaceutical agent comprising (a) calcitriol; which reduces the rateof spine fractures and increases bone density; (b) alendronate, a boneresorption inhibitor, as active ingredients in an optimal mixing ratioto exert the greatest therapeutic effect; and (c) adequate amount ofadditives such as resorption fortifier of alendronate, so that theresulting composition can reduce hypercalceria caused by calcitriol,compensate the inhibitory activity of bone remodeling caused byalendronate due to calcitriol, improve drug compliance usuallyaccompanied because of the difficulty in administration and the usualside effects in esophagus, thus effectively preventing the occurrence ofosteoporosis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows microsopic views (×40) of trabeculae bone of tibia aftertwo months of administration of alendronate and calcitriol, administeredeither independently or as a concomitant administration of bothingredients, following ovariosteresis.

FIG. 2 is a graph that shows the area of trabecular bone observed byusing an image analyzer.

FIG. 3 is a graph that shows the concentration of calcium observed byusing Technicon autoanalyzer.

FIG. 4 is a graph that shows the concentration of calcium/creatinine inurine observed by using a calcium kit (Youngdong Pharmaceutical Co.,Ltd., Korea).

FIG. 5 is a graph that shows the concentration of osteocalcin in serumobserved by a kit using RIA method.

FIG. 6 is a graph that shows the activity of alkaline phosphataseobserved by using Technicon autoanalyzer.

FIG. 7 is a graph that shows the concentration of type I collagentelopeptide (NTx) in urine observed by using Technicon autoanalyzer,which is used as an index for the rate of bone resorption.

For the FIGS. 1-7, A-F represent groups of experimental Sprague-Dawleywhite rats treated as shown below.

A: No Treatment

B: Single Treatment with Excipients

C: Single Treatment with Calcitriol (0.1 μg/kg)

D: Single Treatment with Alendronate (1.0 mg/kg)

E: Composite Treatment 1 [Calcitriol (0.1 μg/kg)+Alendronate (0.5mg/kg)]

F: Composite Treatment 2 [Calcitriol (0.1 μg/kg)+Alendronate (1.0mg/kg)]

G: Composite Treatment 3 [Calcitriol (0.1 μg/kg)+Alendronate (2.0mg/kg)]

FIG. 8 is a graph that shows the change in body weight of Sprague-Dawleywhite rats observed for 2 weeks which were orally treated with acomposite pharmaceutical agent containing both calcitriol andalendronate.

FIG. 9 is a graph that shows the dietary intake of Sprague-Dawley whiterats observed for 2 weeks, which were orally treated with a compositepharmaceutical agent containing both calcitriol and alendronate.

For the FIGS. 8-9, 1-8 represent groups of experimental Sprague-Dawleywhite rats treated as shown below.

1: Calcitriol (125 μg/kg)+Alendronate (1250 mg/kg)

2: Calcitriol (100 μg/kg)+Alendronate (1000 mg/kg)

3: Calcitriol (75 μg/kg)+Alendronate (700 mg/kg)

4: Calcitriol (50 μg/kg)+Alendronate (500 mg/kg)

5: Calcitriol (25 μg/kg)+Alendronate (250 mg/kg)

6: Calcitriol (5 μg/kg)+Alendronate (50 mg/kg)

7: Calcitriol (1 μg/kg)+Alendronate (10 mg/kg)

8: Calcitriol (0.2 μg/kg)+Alendronate (2.0 mg/kg)

FIG. 10 is a microscopic view (H & E×100) that shows the result oftissue findings of the spleen of Sprague-Dawley white rats orallytreated with a composite pharmaceutical agent containing 100 μg/kg ofcalcitriol and 1000 mg/kg of alendronate.

FIG. 11 is a graph that shows the change in body weight ofSprague-Dawley white rats observed for 7 weeks, which were orallytreated with a composite pharmaceutical agent containing both calcitrioland alendronate.

FIG. 12 is a graph that shows the dietary intake of Sprague-Dawley whiterats observed for 7 weeks, which were orally treated with a compositepharmaceutical agent containing both calcitriol and alendronate.

For the FIGS. 11-12, 1-4 represent groups of experimental Sprague-Dawleywhite rats medicated as shown below.

1: Calcitriol (25 μg/kg)+Alendronate (250 mg/kg)

2: Calcitriol (5 μg/kg)+Alendronate (50 mg/kg)

3: Calcitriol (1 μg/kg)+Alendronate (10 mg/kg)

4: Calcitriol (0.2 μg/kg)+Alendronate (2 mg/kg)

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a pharmaceutical composition for thetreatment of metabolic bone disease characterized by having calcitrioland alendronate as two active ingredients wherein said pharmaceuticalcomposition contains 1,000 to 50,000 parts by weight of alendronate per1 part by weight of calcitriol.

The present invention also relates to a process of preparing saidpharmaceutical composition containing calcitriol and alendronate asactive agents comprising

a) a process of obtaining calcitriol granules;

by adding mannitol to a mixture of calcitriol and ethanol,

subsequent adding ethanol, antioxidant and binder to said mixture,

pulverizing said mixture into 14-35 mesh size and drying said pulverizedmixture;

b) a process of obtaining alendronate granules;

by mixing mannitol, resorption fortifier and alendronate,

subsequently adding ethanol and binder to said mixture,

pulverizing said mixture into 14˜35 mesh size and drying said pulverizedmixture;

c) a process of preparing tablets;

by mixing said calcitriol granules obtained from said step a) and saidalendronate granules obtained from said step b) with 1:1 part by weightof mixing ratio followed by subsequent adding of a disintegrating agentand a lubricant to said mixture and tableting the mixture.

This invention is explained in more detail by the following examples,but they should not be construed as limiting the scope of thisinvention.

The present invention relates to a method of manufacturing a compositepharmaceutical agent comprising calcitriol and alendronate as its twoactive ingredients at its optimal mixing ratio, wherein the formeringredient is known to be effective in reducing spinal fractures andincreasing bone density while the latter effective in inhibiting boneresorption, followed by the addition of resorption fortifier.

The above two active ingredients are further elucidated as follows.

First, calcitriol expressed as 1,25-(OH)₂-D₃, is one of active vitaminD₃ derivatives involved in calcium transport in intestinal tract and isalready known that it can activate intestinal calcium absorption as wellas it can reduce the secretion of parathyroid hormone thus alleviatingthe bone loss of menopausal women suffering from osteoporosis [Aloia J.F., Am J Med (1988), 84, 401-8]. In addition, calcitriol is also knownto activate the production of osteocalcin, a bone-specific proteininvolved in mineralization of bones with respect to osteoblats [Price P.A., Calcif Tissue Int (1990) Apr., 46(4), 270-9]. However, calcitriol asa pharmaceutical agent also has a problem because it can often causehypercalcemia when administered alone and therefore there have been manylines of studies in order to resolve this drawback.

Second, alendronate, expressed as4-amino-hydroxybutylidine-1,1-bisphosphonate, is a kind ofbisphosphonates developed as a therapeutic agent for the treatment ofosteoporosis and is known to prevent bone loss by inhibiting theactivity of osteoclasts thus helping to improve the bone density. Infact, clinical studies showed that there was about 2-3% increase in bonedensity. For example, a clinical study which was implemented on 994post-menopausal women from 16 different countries showed that there wasa gradual increase in mineral density of spinal bone and bones of lowerlimbs. Also, the rate of occurrence of new spinal fracture in a groupmedicated with alendronate was reduced by about 48% as compared to thatof a placebo group.

In general, the mineral density of a bone is closely associated withbone density and thus the rate of occurrence of bone fracture becomesdrastically decreased as the mineral density becomes higher. Whenalendronate was administered 10 mg/day for 3 years (10 mg/day ofalendronate is thought to have the most effective therapeutic effect[Liberman, N. Engl. J. Med. (1995), 333, 1437-1443], the mineraldensities of spinal bone and bones of lower limbs were increased by 8.2%and 7.2%, respectively, whereas the mineral density on a placebo groupwas dropped by 0.7% during the same period of administration. However,alendronate is shown to adversely affect the normal cycles of bonemetabolism by reducing the rate of bone remodeling and is therefore acomposite pharmaceutical agent necessitated to compensate this problem.

Since alendronate is able to prevent hypercalcemia, which is known asthe most serious side effect caused by calcitriol, it has beenpostulated that combined administration of both alendronate andcalcitriol would be able to resolve hypercalcemia and also enable toadminister optimal amount of calcitriol, which has been limited itsdaily dosage due to its narrow safety margin. Moreover, the antagonisticactivity of alendronate against bone remodeling will be also compensatedby the presence of calcitriol.

As a way to develop a most effective composite pharmaceutical agent forthe treatment of post-menopausal women, experimental animals were firstinduced to develop osteoporosis in the present invention and then theywere placed under numerous experiments of administering variousconcentrations of mixtures of calcitriol and alendronate resulted fromvarious mixing ratios between calcitriol and alendronate, and finalizedtheir optimal mixing ratio which can not only effectively preventosteoporosis without retarding the rate of bone remodeling but also cancontrol the calcium concentration in blood to be kept at normal level.The results showed that the optimal concentration ratio between the twoingredients of a pharmaceutical agent was to contain 1,000-50,000 partsby weight of alendronate with respect to 1 part by weight of calcitriol.If the amount of alendronate contained in said pharmaceutical agent isless than 1,000 parts by weight per 1 part by weight of calcitriol, theresulting plasma calcium concentration will be increased and there willbe a high risk of causing hypercalcemia when administered with thepharmaceutical agent. On the other hand, the amount of alendronatecontained in said pharmaceutical agent is more than 50,000 parts byweight per 1 part by weight of calcitriol, it will adversely affect thenormal cycles of bone metabolism by reducing the rate of boneremodeling.

The pharmaceutical agents according to the present invention can beprepared in various dosage forms such as naked tablets, enteric coatedtablets, granules, enteric coated granules, capsules, and enteric coatedcapsules. Further, it is recommended to use each ingredient being in therange of 0.1-5.0 μg for calcitriol and 1-50 mg for alendronate per eachdosage unit.

Additives were selected after a series of experiments from those whichhave been already shown their effectiveness in preparing pharmaceuticalagents. Adequate amount of selected additives were then added to each ofthe above two active ingredients respectively to prepare two preliminarypreparations and then they were combined together to finally form acomposite pharmaceutical agent. Thus obtained composite pharmaceuticalagent exhibited much improved drug compliance compared to those ofconventional pharmaceutical drugs which have relatively low drugcompliance mainly due to the difficulty in administration and the sideeffects in esophagus, thus effectively preventing the osteoporosis.

The specific features of the manufacturing processes employed in thepresent invention are as follows.

Process A

In the present invention, mannitol is added to a mixture of calcitrioland ethanol and then mixed, and then ethanol, an antioxidant and abinder are added to the mixture and mixed. The resulting mixture is thenpulverized into a size of 14-35 mesh and then dried at 30-50° C. togenerate calcitriol granules. Here, if the size of the mixture is lessthan 14 mesh then the resulting calcitriol granules becomenon-homogeneous while if the size is larger than 35 mesh the productionyield becomes low and the processability in tableting also becomesworsened.

Process B

In a separate step apart from the above process A, alendronate, mannitoland an resorption fortifier are mixed together, and then added withethanol and a binder.

The resulting mixture is then pulverized into a size of 1435 mesh andthen dried at 30-50° C. to generate alendronate granules. Here, if thesize of the mixture is less than 14 mesh then the resulting alendronategranules become non-homogeneous while if the size is larger than 35 theproduction yield becomes low and the processability in tableting alsobecomes worsened.

Process C

The calcitriol granules and alendronate granules obtained from the abovementioned process A and B, respectively, are mixed together, added witha disintegrating agent and a lubricant, and the resulting mixture istableted to finally produce the composite pharmaceutical agent of thepresent invention.

The additives used in the present invention are described hereunder.

First, mannitol is used as an excipient in the above processes A and B,and the amount of mannitol used in the processes A and B is limited sothat the amount be 50-60 weight % and 40-60 weight % of the total amountof mannitol contained in the final pharmaceutical composition,respectively.

The reasons of limiting the amount of mannitol are as follows. Themiscibility test between calcitriol and alendronate showed that themixture of the two components resulted in deterioration of the stabilityof calcitriol. However, in a compatibility test, when calcitriol wasmixed with other excipients before mixing with alendronate thecalcitriol in the resulting mixture remained stable. Therefore, in orderto avoid the direct contact between calcitriol and alendronate, theinventors of the present invention first diluted calcitriol andalendronate independently with mannitol to produce their granules andthen mixed them thereafter. With respect to the amount of mannitol usedin preparing said granules, it was found that the amount of mannitol isimportant in maintaining homogeneity of said granules.

When both granules of calcitriol and alendronate were prepared by using50% of the total mannitol, respectively, there were observed differencesin the resulting granules in terms of specific volume and the tabletssubsequently obtained after mixing and tableting of said granules werenot uniform in the amount of said ingredients. Therefore, numerousexperiments were conducted to find an optimal amount of mannitol toproduce a homogeneous granule as well as to contain an equal amount ofeach given ingredient; as a result, it was found that using about 50-60%of total mannitol is suitable for the preparation of calcitriolgranules, and preferably 52%, while about 40-60% of total mannitol isrecommended for the preparation of alendronate granules, and preferably48%. The above two granules were then adjusted to have equal amountbefore mixing them together.

The examples of excipients that can be used in the present inventioninclude white sugar, cellulose and lactose as well as mannitol, andpreferably D-mannitol. These excipients are recommended to use 10-98weight %, and preferably 7097 weight %. If the amount of mannitol addedis off the above range then the processability in tableting becomesworsened.

Antioxidants that can be used in the present invention are one or moreselected from the group consisting of butylated hydroxytoluene(BHT),(butylated hydroxyanisole(BHA), DL-alpha-tocopherol and lecithin, andpreferably butylated hydroxytoluene(BHT) and butylatedhydroxyanisole(BHA). These antioxidants can be used 0.001-10 weight % ofthe total pharmaceutical composition, and preferably 0.01-1.0 weight %.If the amount of antioxidants is off the above range then there will beraised a problem of calcitriol stability.

A binder can be selected from the group consisting of pyrrolidone,hydroxypropyl methyl cellulose, hydroxypropyl cellulose and carboxymethylcellulose sodium.

The recommended amount of the binder as an additive is to use 0.1-20weight % of the total pharmaceutical composition, and preferably 1-10weight %. If the amount of a binder is off the above range then theprocessability in tableting will be deteriorated due to non-uniformgranular distribution.

A disintegrating agent can be selected from the group consisting ofcroscarmellose sodium, low-substituted hydroxypropyl cellulose andcarboxy methylcellulose calcium, and the recommended amount of thedisintegrating agent as an additive is to use 0.1-20 weight % of thetotal pharmaceutical composition, and preferably 1-10 weight %. If theamount of a disintegrator is off the above range then there will be aproblem in the disintegration of tablets.

Examples of lubricants include calcium stearate, magnesium stearate andtalc, it is recommended to use 0.1-20 weight % of the totalpharmaceutical composition, and preferably 1-10 weight %. If the amountof a disintegrating agent is off the above range then there will be aproblem in processability of tableting as well as in the assay of activeingredients.

Sodium lauryl sulfate can be used as a resorption fortifier and itsrecommended amount of use is 0.01-10 weight % of the totalpharmaceutical composition, and preferably 0.1-1 weight %.

In addition, additives for a sustained release can be selected from thegroup consisting of hydroxypropyl methyl cellulose, methyl cellulose,hydroxypropyl cellulose and ethyl cellulose.

The pharmaceutical components according to the present invention can beprepared as enteric coated tablets by coating with an enteric coatingsolution and they can be also prepared as capsules by filing a givenpharmaceutical composition into conventional type of capsules. Theenteric coating solution consists of an enteric coating agent, aplasticizer and a solvent. Examples of the enteric agents arehydroxypropyl methyl cellulose phhalate, metacrylic acid polymer andcellulose acetate phthalate, and examples of platicizers includetriethyl citrate, polyethylene glycol and propylene glycol. Solventsused in the present invention are two or more selected from the groupconsisting of methylene chloride, ethanol, acetone and distilled water.

Thus prepared pharmaceutical agents have shown superior therapeuticeffects in treating metabolic bone disease such as osteoporosis, Paget'sdisease, rachitis, osteomalacia, renal osteodystrophy of renal failurepatients, hypoparathyroidism and hyperparathyroidism.

This invention is further illustrated by the following examples.However, these examples should not be construed as limiting the scope ofthis invention in any manner.

REFERENTIAL EXAMPLE

Optimal concentration ratio was calculated as follows by which not onlythe progress of osteoporosis can be effectively prevented but the plasmacalcium concentration as well as the amount of calcium excretion viaurine can be maintained at a normal level while not inhibiting the rateof bone remodeling.

1) Induction of Osteoporosis

Twelve week old matured female Sprague-Dawley white rats were suppliedfrom Daehan Biolink Co., Ltd. (Seoul, Korea) and raised in a dean roomof an animal experiment laboratory in a medical school of Seoul NationalUniversity. The white rats were given a week to adapt by feeding themwith normal feedstuff (Purina, chow diet) and then they were dividedinto 6 different groups wherein each group consisted of 10 white ratswith similar weight distribution, and ovariostereses were performed toall of them. The white rats were allowed to freely drink water and havefood under a standardized condition of 22° C., 50% humidity, 12 hdark/light cycle.

2) Medication

After the ovariostereses, the white rats were rested for 2 days and thenorally administered in the morning for 5 days per week for the durationof 2 months with a medication which contains certain amount of eithersingle or combination of both calcitriol and alendronate, which wereindependently dissolved in 0.5 mL of purified palm oil and physiologicalsaline, respectively. The amount of medication administered in eachgroup is shown in the following table 1 and the control group wasadministered only with equal amount of palm oil and physiological salineused in the experimental groups.

TABLE 1 Phys- Purified iological Calcitriol Alendronate Palm oil salineClassification (μg/kg) (mg/kg) (mL) (mL) Control Group — — 0.5 0.5 Groupadministered with 0.1 — 0.5 0.5 calcitriol Group administered with — 1.00.5 0.5 alendronate Group administered 1 0.1 0.5 0.5 0.5 with bothcalcitriol 2 0.1 1.0 0.5 0.5 and alendronate 3 0.1 2.0 0.5 0.5

3) Collecting Samples and Pretreatment

After 2 months of the onset of the above medications, 24 h urine sampleswere collected from the white rats by using a metabolic cage,centrifuged and then stored at −70° C. The white rats under experimentwere fasted overnight decapitated, and their blood samples werecollected. White rats sera were then obtained by centrifuging thoseblood samples and were stored at −70° C. Tibiae were separated byablation and fixed with 4% formalin at pH 7.4.

4) Statistic Data and the Evaluation

The result of each experiment was represented by mean value±standarddeviation, and the significance was determined by using ANOVA, Duncanmultiple range test at the level of P<0.05.

Experimental Example 1 Bone Histomorphometry

For the decalcification of tibiae fixed on the above referentialexample, each tibia was placed in a 10% nitric acid solution for 6 h andthen dehydrated by passing through 10 different alcohol concentrationsranging from 80% to 100%. Then, it was embedded twice in xylene andparaffin, respectively, cut proximal tibia using a bone-cutting microsawinto 4 cm thick pieces and dyed with hematoxyleneasin. The aboveproximal tibiae were observed under 42× magnification of a microscopeand the ratio between the area of trabecular bone within a standard areaof μm² and the standard area was calculated after measuring the area oftrabecula by using quantitative image analysis System. The results ofbone histomorphometry are as follows.

About 2-3% of cortical bone in humans is replaced annually; in contrast,trabecelar bone is replaced by a much faster rate of 25% per year andthus the rate of development of trabecular bone can be used as a directas well as an exact W of discerning the state of bone health, and itthus has been widely used in animal experiments. In the presentexperiment, alendronate and calcitriol were administered either singlyor in combination for 2 months after an ovariosteresis and microscopicobservation of the trabecular bone of tibia showed that the area oftrabecular bone was much reduced as compared to that in a normal group(see FIG. 1). Observations of the experimental groups using the imageanalyzer revealed that the area of trabecular bone was drasticallyincreased in a group treated with alendronate alone; groups 1, 2 and 3,which were treated with a composite drug, whereas it was not increasedin a group medicated with calcitriol alone (see FIG. 2). For example, interms of the area of trabecular bone, the group treated with a compositedrug containing alendronate and calcitriol showed the same effect as inthe group treated with alendronate alone, and also there was nodifference among the groups 1, 2 and 3, which were treated with acomposite medication, respectively.

Experimental Example 2 Biochemical Test

Reagents used were those obtained from the referential example above.

1) Measurement of Serum Calcium Concentration

The serum calcium concentration was measured by using Techniconautoanalyzer and the result is shown below.

There was a significant increase in serum calcium concentration in thegroup treated with calcitriol alone while the group treated withalendronate alone showed a significant decrease in serum calciumconcentration. This is because calcitriol increases calcium absorptionin intestine while alendronate inhibits bone resorption by reducingparathyroid hormone, which then prevents the release of calcium from abone into a blood stream and inhibits the synthesis of calcitriol[1,25-(OH)₂-D₃] in kidney thus decreasing the calcium absorption inintestine. Therefore, the serum calcium concentrations in groups 1, 2and 3 administered with a composite drug were maintained at theintermediate levels in between the concentration of the group treatedwith calcitriol alone and that of the group treated with alendronatealone, as expected (see FIG. 3).

2) Measurement of Calcium Excretion in Urine

In order to study the effect of medication on the relationship betweenthe serum calcium concentration and the resulting calcium excretion inurine, the concentration ratio of calcium/creatine of a 24 h urinesample was measured by using a calcium kit (Yeongdong PharmaceuticalCo., Ltd., Korea) and the result is shown in FIG. 4. According to theFIG. 4, the calcium excretion was increased markedly when medicated withcalcitriol while there was no difference observed when medicated withalendronate. The amount of calcium excretion increased in groups 1 and 3with a composite drug containing both calcitriol and alendronate,whereas that of group 2 remained at the level comparable to that of acontrol group (FIG. 4).

3) Measurement of Bone Remodeling Index

The activity level of serum alkaline phosphatase was measured by usingTechnicon autoanalyzer and the concentration of osteocalcin was measuredby using a kit (OSCA test, Brahams, Berlin, Germany) which uses RIAmethod.

As an index of bone remodeling rate, the concentration of serumosteocalcin and the activity of alkaline phosphatase were measured.There was a slight increase in both the concentration of osteocalcin andthe activity of alkaline phosphatase when medicated with calcitriolalone while they were decreased when medicated with alendronate,although not to a significant level (FIGS. 5 & 6). There was no changeobserved in concentration of osteocalcin in all the groups treated witha composite medication. The activity of alkaline phosphatase decreasedsignificantly in the group 3 with a composite medication, however, thegroups 1 and 2 did not show any noticeable change in the activity ofalkaline phosphatase thus implying that the rate of bone remodeling didnot decrease in groups 1 and 2.

4) Measurement of Bone Resorption Index

The ratio of NTx/creatine in urine was calculated by measuring theconcentration of N-telopeptide (NTx) of a 24 h urine sample, aby-product of type I collagen, using a kit (Osteomark, Ostex, USA). Theresult showed that there was no significant change in the rate of boneresorption due to medications (see FIG. 7).

From the above experimental examples 1 and 2, the group 2 with acomposite medication (0.1 μg/kg of calcitriol+1.0 mg/kg of alendronate)was shown to be the most optimal concentration ratio for a compositepharmaceutical preparation because the progress of osteoporosis waseffectively prevented without inhibiting the rate of bone remodeling,and also the serum calcium concentration and the calcium excretion inurine were maintained at a normal level. Thus obtained compositepharmaceutical preparations were evaluated for their safety according tothe standard acute and subacute toxicity tests described in Korea Food &Drug Administration 9856 issued on Apr. 29, 1998 as in the followingexperimental examples 3 and 4 and their safety was accordinglyconfirmed.

Experimental Example 3 Acute Toxicity Test of a Composite PharmaceuticalAgent containing Both Calcitriol and Alendronate

The toxicity of a single dose of a composite pharmaceutical agentcontaining calcitriol and alendronate via oral administration onexperimental animals was examined to evaluate the safety of the abovecomposite pharmaceutical agent. In this experiment, the above compositepharmaceutical agent was administered on Sprague-Dawley white rats tostudy the acute oral toxicity by means of LD₅₀, general conditions, bodyweight and the amount of dietary intake, and was examined further by anautopsy.

Nursing Conditions

One hundred eighty of 45 week old Sprague-Dawley white rats (90 malesand 90 females) were raised in a SPF room designed by Techniplast Co. inItaly, located annexed to the animal care center in the Department ofPharmacology of Choong-Ang University in Korea under the conditions of23±2° C., relative humidity of 60+29%, 13-15 times of airventilations/h, illumination of 12 h/day (from 7 a.m.-7 p.m.). The aboveexperimental white rats were placed in a cage and allowed to freelyintake solid feedstuff (Hanil Feed: Greenpia Co., Ltd., Korea)sterilized by an X-ray irradiation and drinks (heat-sterilized tapwater).

Preparation of Sample Reagents

Calcitriol stored in a light-resistant container in a −20° C. freezerwas weighed for 1.0 mg and added into a brown flask. 1.0 μg/mL and 10μg/mL solutions were freshly prepared by adding a solvent (100% ethanol:MCT=5:95) when needed, and mixed with a 0.5% CMC-Na aqueous solution,where alendronate monosodium was already suspended.

Determination of the Amount of Medication

The mixing ratio between the amount of medication for calcitriol andalendronate was determined to be 0.5 μg:5 mg based on the amountadministered to humans. Animals were proportionally divided into 8different groups based on the values of LD₅₀ and the total of 9 groupsincluding a control group were determined as shown in the table 2.Medication was orally administered after making the volume totalling 1mL. Based on the relationship between the human body weight and theamount administered to white rats, the minima doses were set to be 0.2μg/kg and 2.0 mg/kg for calcitriol and alendronate, respectively.

TABLE 2 Group 1 2 3 4 5 6 7 8 Control Calcitriol 125 100 75 50 25 5 10.2 0.0 (μg/kg) Alendronate 1250 1000 750 500 250 50 10 2.0 0.0 (mg/kg)

Experimental Items

1) Measurement of LD₅₀

In order to measure the values of LD₅₀ f both male and female whiterats, animals under experiment were divided into 8 medication groups anda control group wherein each group consisted of 10 white rats and wasorally administered. Then, the number of animals which were dead withintwo weeks after the medication were counted and their LD₅₀ values werecalculated by using Brehrens-Karber method.

The resulting LD₅₀ of white rats administered with the composite drugcontaining calcintriol and alendronate are as follows.

The moralities for both male and female white rats, treated with asingle oral administration with the composite medication consisting ofcalcitriol and alendronate (1 μg/kg: 10 mg/kg), are shown in thefollowing table 3.

TABLE 3 Oral Administration (calcitriol + alendronate) Mortalitycalcitriol(μg/kg) alendronate(mg/kg) Female Male 125 1250 10/10  10/10 100 1000 9/10 9/10 75 750 6/10 5/10 50 500 0/10 0/10 25 250 0/10 0/10 550 0/10 0/10 1 10 0/10 0/10 0.2 2.0 0/10 0/10

The values of LD₅₀ of the composite drug of calcitriol and alendronatewere calculated according to Brehrens-Karber method. The result showedthat the LD₅₀ of the male rat was 77.5 μg/kg for calcitriol and 775mg/kg for alelidronate, whereas that of female rat was 75.0 μg/kg forcalcitriol and 750 mg/kg for alendronate.

2) Observation of Normal State

Observations were made daily for all the white rats under experiment forthe duration of 2 weeks in such a manner that each mouse was observedevery 30 min for the first 6 hr after the medication and then observedonce a day at predetermined time from the second day. The results ofobservations of white rats at normal state are as follows.

For the first 6 hr after the medication, the group of female white ratsmedicated with more than LD₅₀ showed a peculiar behavior that theyscratched their heads using their legs and the behavior turned out to bea symptom due to the esophagogastric stimulation. Then, those white ratsshowed convulsion and dyspnea within 1 hr from the medication. Theyshowed a decrease in autopathy after 2 hr from the medication, a lightdegree of behavioral flexibility, maintained lethargic state, and hadrespiratory stress. Almost all the white rats medicated with not lessthan 100 μg/kg of calcitriol and 1000 mg/kg of alendronate died within24 hr of the medication. In the group of white rats medicated with 75μg/kg of calcitriol and 750 mg/kg of alendronate, 60% of female and 50%of male were dead while in the group medicated with 50 μg/kg ofcalcitriol and 500 mg/kg of alendronate were all alive. In the group ofwhite rats medicated with 50 μg/kg of calcitriol and 500 mg/kg ofalendronate, 3 out of 10 showed the above-mentioned esophagogastricstimulation symptom but they recovered from the symptom on the first dayof the convalescence.

3) Measurement of Body weight and Food Intake

All the white rats under experiment were daily measured for theirweights and their food intakes at predetermined time before and aftereach medication for 2 weeks. The results showed that there was nosignificant change in male and female white rats except those which weredead in food intake as well as in body weight (FIGS. 8-9).

4) Autopsy

Gross findings were obtained from the dead white rats as well as fromall the live white rats after killing them by anesthetizing with ether.Abnormal organs or tissues detected by naked eyes were fixed with 10%neutral formalin and then made into standard tissue samples. They werethen dyed with hematoxylene and eosin to obtain tissue findings and theresults are as follows.

The autopsies of white rats treated with a composite drug containing notless than 75 μg/kg of calcitriol and 750 mg/kg of alendronate showed noabnormal findings except hemostasis of spleen. Autopsies performed forcontrol white rats and those alive at completion of convalescence of 14days after treating with a composite drug containing calcitriol andalendronate, and there were no abnormal findings except minorhypertrophy of spleen. Tissue autopsies of white rats which were founddead after treating with 100 μg/kg of calcitriol and 1,000 mg/kg ofalendronate showed that there were serious hemostasis and hemorrhage ofspleen (FIG. 10).

Experimental Example 4 Subacute Toxicity Test of a CompositePharmaceutical Agent Containing Both Calcitriol and Alendronate

Subacute toxicity for a 5 week repetitive dose of a compositepharmaceutical agent containing calcitriol and alendronate via oraladministration on experimental white rats was examined to evaluate thesafety of the above composite pharmaceutical agent. White rats underexperiment were examined once per each day for the duration of 5 weeksof repetitive oral medications and for the duration of 2 weeks ofconvalescence. In order to evaluate the subacute toxicity of thecomposite pharmaceutical agent, experiments such as normal state, changein body weight, dietary intake, ophthalmologic examination, acoumetry,urine test, hematological test, hematochemical test, and pathologicaltest were performed after treating with a given medication.

Experimental Animals and Nursing Conditions

Sprague-Dawley white rats were raised under the same condition asdescribed in the acute toxicity test in the Experimental Example 3, withthe exception that they were placed into a metabolic cage one day priorto the completion of the experiment. Each experimental group consistedof 20 males and 20 females, and they were treated for 5 weeks. After 2weeks of convalescence, 10 males and 10 females were selected from eachgroup and were examined.

Preparation of Sample Reagents & Determination of the Amount ofMedication

Sample reagents were prepared the same as described in the acutetoxicity test in Experimental Example 3. The mixing ratio between theamount of medication for calcitriol and alendronate was determined to be0.5 μg: 5 mg based on the amount of medication for humans, LD₅₀ valuesalready reported, and the amount of medication known to be effective inclinical treatment and prevention of osteoporosis. Five groups wereselected via azetropy 5 with 4 different medication groups and a controlgroup as shown in the table 4.

TABLE 4 Group 1 2 3 4 Control Calcitriol(μg/kg) 25 5 1 0.2 0.0Alendronate(mg/kg) 250 50 10 2.0 0.0

Experimental Items

1) Mortality Examination

No mortality was observed but both male and female white rats treatedwith a composite drug containing calcitriol and alendronate were allalive both at the time of completion of medication and at convalescence.

2) Observation of Normal State

Normal state of white rats were observed (a) 3 times per each day duringtreatment; before medication, immediately after medication, and 2 hrafter medication, (b) 2 times per each day during holidays; beforemedication and immediately after medication, and (c) once per each dayduring convalescence.

Esophagogastric stimulating symptom and lethargic state were observed upto day 3 after medication but those symptoms disappeared after day 5.All the other experimental groups did not show any particular change innormal state that appears to be due to the medication.

3) Change in Body Weight and Water Intake

Body weight was measured once every 3 days during the medication andthrough the convalescence. The accumulated amount of water intaken for aperiod of 7 days was measured once per week and the amount of dailyintake per each mouse was estimated from this. The result showed thatthere was no significant change in body weight (FIG. 11) nor in waterintake (FIG. 10) in all the experimental groups treated with thecomposite pharmaceutical agent containing calcitriol and alendronateuntil the completion of medication and convalescence.

4) Ophthalmologic Autopsy

Ophthalmologic autopsies were executed for all the white rats under theexperiment during the inspection, and on the 5^(th) week of theexperiment only 5 males and 5 females selected from each group wereexamined. While observing the external ophthalmologic state atropine wasinstiliated and anterior segment of eye, intermediate transmitting bodyof eye, and fundus of eye were examined. When there was no change due toa given medication observed by the examination on the 5^(th) week, thesubsequent examination at convalescence was omitted. The result showedthat there was no significant ophthalmologic change in all theexperimental groups treated with the composite pharmaceutical agent ofcalcitriol and alendronate until the completion of medication andconvalescence.

5) Acoumetry

Outreflex was examined on the 5^(th) week of medication by means of aGaidan-Boizuru machine (about 600 Hz) for 10 males and 10 females fromeach experimental group. When there was no change due to a givenmedication observed by the examination on the 5^(th) week, thesubsequent examination at convalescence was omitted. The result showedthat there was no significant acoumetric change in all the experimentalgroups treated with the composite pharmaceutical agent of calcitriol andalendronate until the completion of medication and convalescence.

6) Urine Test

On the 5^(th) week of medication and on the 2^(nd) week ofconvalescence, 10 males and females were selected from each experimentalgroup and were forced to fast but allowed to drink water, and urinesamples were collected from them after 4 hr of fasting. Then they wereallowed to freely intake both food and water, and urine samples werecollected from them after 20 hr of their free diet. The urine samplesobtained from the first 4 hr were analyzed with respect to leucocytes,nitrite, urobilinogen, pH, proteins, ketone bodies, urine sugar, occultblood, specific gravity, biliruibn (AMES urine test paper, N-NultistixSG-L: Japan Bayer, Samkong Co., Ltd.), colors and sediments. Urinesamples obtained from 20 hr were used to estimate Na, K and Cl (totalquantity measurement). The result showed that there were no significantchange in urines obtained from all the experimental groups treated withthe composite pharmaceutical agent of calcitriol and alendronate untilthe completion of medication and convalescence but they all stayedwithin the normal range.

7) Hematological Examination

White rats were fasted overnight for about 16 hr before killing and theywere dissected along the median line after anesthetizing with ether.Blood samples were collected from the abdominal arteries and stored invacutainers (Bechon Dickinson Vacutainer Systems Europe England)containing anticoagulant (7.5% EDTA-2K). The blood samples were thenused to analyze red blood cell (RBC) count (detection of change inelectric resistance), amount of hemoglobin (cyanomenohemnoglobinmethod), hematocrit value (estimate RBC count and mean hematocritvolume), mean corpuscular volume (detection of change in electricresistance), mean corpuscular hemoglobin (estimate from the amount ofhemoglobin and hematocrit value), platelet count (detection of change inelectric resistance), (Abnormality Automatic Analyzing InstrumentCell-Dyne 3000, Abbot Co.), reticulocyte (Brecher method) anddifferential white count (May-Giemsa method). Here, blood plasmaobtained by centrifugation (10 min at 3,000 rpm) of blood samplestreated with 3.8% sodium citrate was used to estimate theabove-mentioned tests by means of prothrombin time (Quick method) andthe results are as follows.

At Completion of Medication

There was no significant change observed in all the experimental groupstreated with the composite pharmaceutical agent of calcitriol andalendronate.

At Completion of Convalescence

There was no significant change observed in all the experimental groupstreated with the composite pharmaceutical agent of calcitriol andalendronate.

8) Hematochemical Examination

Blood samples were collected from abdominal veins as in the case withhematological examination and treated with heparin. Blood sera wereobtained by centrifugation (10 min at 3,000 rpm) of the blood samplesand were used to analyze GPT and GOT (Abnormal UV rate method,Abnormality Automatic Analyzing Instrument, CH16, Texas InternationalINC.), ALP (Bessey-Lowry method), total cholesterol (CEH-COD-PODmethod), total bilirubin, blood glucose (hexokinase PD method),urea-nitrogen (UreaGL PC method), inorganic phosphorous (molybdic acidmethod) and total protein (Biuret method) (Abnormal UV rate method,Abnormality Automatic Analyzing Instrument, Monach, InstrumentationLaboratory). Reagents used in the above examinations were purchased fromStanbio Laboratory Inc. (Texas, USA) and the results are as follows.

At Completion of Medication

There was a little increase in GPT activity in male white rats treatedwith 25 μg/kg of calcitriol and 250 mg/kg of alendronate, and thosetreated with 5 μg/kg of calcitriol and 50 mg/kg of alendronate. GFPactivity was increased in all the experimental white rats. However,there was no significant dosage-dependent change and the level of GTPactivity was relatively high even in a control group treated withsolvent thus implying that the little increase in GTP activity was dueto a light hematolysis incurred while collecting blood samples. Thelevel of blood glucose was increased a little in all the male and femaleexperimental white rats, however, there was no significant dependencyobserved on the volume. There was a little increase in a control grouptreated with solvent and urinal glucose was not detected in urines thussuggesting that the little increase in the level of glucose was not dueto diabetes but a temporary increase due to a stress. Therefore, it canbe concluded from the above results that there was no significantdosage-dependent change in both experimental groups and a control groupdue to medications with the exceptions of a little increase in GOTactivity and glucose level.

At Completion of Convalescence

The GPT activity in male white rats became normal at completion ofconvalescence treated with 25 μg/kg of calcitriol and 250 mg/kg ofalendronate, and those treated with 5 μg/kg of calcitriol and 50 mg/kgof alendronate. GOT activity became lower in all the experimental groupsas compared to that at completion of medication. However, there was nosignificant dosage-dependent change and the level of GTP activity wasrelatively high even in a control group treated with solvent thusimplying that the little increase in GTP activity was due to a lighthematolysis incurred while collecting blood samples. Therefore, it canbe concluded from the above results that there was no significantdosage-dependent change in both experimental groups and a control groupdue to medications with the exceptions of a little increase in GOTactivity and glucose level as is the case with at completion ofmedication.

9) Autopsy

Gross findings were obtained from the dead white rats as well as fromall the live white rats after killing them by anesthesizing with ether.Abnormal organs or tissues detected by naked eyes were fixed with 10%neutral formalin and then made into standard tissue samples. They werethen dyed with hematoxylene to obtain tissue finding and the results areas follows.

At Completion of Medication

The autopsies of white rats treated with a composite medication of 25μg/kg of calcitriol and 250 mg/kg of alendronate showed a significantlevel of hypertrophy of spleen but there were no abnormal findings inwhite rats treated with less than the above-mentioned dosage. There wereno tissue findings that showed significant changes due to medicationother than spleen.

At Completion of Convalescence

The autopsies of white rats treated with a composite medication of 25μg/kg of calcitriol and 250 mg/kg of alendronate showed a significantlevel of hypertrophy of spleen but there were no abnormal findings inwhite rats treated with less than the above-mentioned dosage. There wereno tissue findings that showed significant changes due to medicationother than spleen.

10) Pathological Examination

Upon collecting blood samples all the experimental white rats werekilled by exsanguinations. After observing the abnormalities of all theorgans by naked eyes, brains, hypothalamus, thyroid gland includingparathyroid, salivary gland (glandular submaxillaris and glandularsubligualis), thymus, heart, lungs including bronchi, liver, spleen,kidney, testicle, adrenal gland, ovary, prostate gland, seminal veside,uterus, and sternum were ablated and weighed, respectively. Theexperimental white rats were fasted on the day of autopsy and therelative weight of each organ mentioned above was estimated to 100 g ofbody weight, respectively. Also, abnormal regions of spinal cord, nervusishiadicus, thoracic aorta, trachea, tongue, esophagus, stomach,duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, andmesenterium were ablated and fixed with 10% formaline with phosphatebuffer, and brains and spinal cords (sternum and femur) were fixed withBouin's solution and embedded with paraffin. Specimens of all thetissues and organs obtained from the white rats during medication andconvalescence all the experimental groups and the solvent control groupwere dyed with hematoxylene and eosin (H & E) and observed under amicroscope. The results showed no apparent changes due to medication

Conclusion from the Experimental Examples 3-4

Male and female Sprague-Dawley white rats were orally administered witha composite drug containing both calcitriol and alendronate with amixing ratio of 1:10,000 either once or repeatedly for the duration of 5weeks for the tests of acute and subacute toxicities and the resultswere as follows.

First, the results of acute toxicity test for the above compositemedication are as follows.

The values of LD₅₀ for the oral medication of the composite drug ofcalcitriol and alendronate were calculated by using Brehrens-Karbermethod. The result showed that the LD₅₀ of the male white rats was 77.5μg/kg for calcitriol and 775 mg/kg for alendronate, whereas that offemale white rats was 75.0 μg/kg for calcitriol and 750 mg/kg foralendronate, respectively. All the experimental white rats were alivewhen treated once orally with the composite medication of 50 μg/kg ofcalcitriol and 500 mg/kg of alendronate and there were no abnormalitiesobserved in normal state except esophagogastric stimulating symptom andlethargy. Body weight, the amount of dietary intake and the result ofurine test were all shown to be normal.

The autopsies of white rats killed by the oral treatment of thecomposite drug of not less than 75 μg/kg of calcitriol and not less than750 mg/kg of alendronate revealed that there were serious hemostasis andhemorrhage of spleen, however, no other particular lesions were observeddue to the medication.

Second, the results of subacute toxicity test for the above compositemedication are as follows.

At completion of Repetitive Medication

All the experimental white rats were alive after repetitive oraltreatment for 5 weeks with the composite medication of 25 μg/kg ofcalcitriol and 250 mg/kg of alendronate and there were no abnormalitiesobserved in normal state, the amount of dietary intake, ophthalmologicalexamination, acoumetric examination, hematological examination andhematochemical examination.

The autopsies of the above white rats revealed that there were noabnormal findings in the weight of each organ except hypertrophy ofspleen, however, there were also observed moderate level of local lungseptum and slight hemostasis in spleen, liver, kidney, andesophagogastric mucosal edema and extravasation of inflammatory cells.However, these lesions were also observed in an experimental grouptreated with a low dosage of composite medication of less than 5 μg/kgof calcitriol and less than 50 mg/kg of alendronate as well as in acontrol group and thus these lesions were not considered due to themedication. There were no abnormal findings in the brain, bone marrow,sexual gland and peptic gland.

At Completion of Convalescence

Experimental white rats at completion of convalescence of 2 weeks afterrepetitive oral administration with composite drug of 25 μg/kg ofcalcitriol and 250 mg/kg of alendronate for 5 weeks were all alive andthere were no abnormalities observed in normal state, the amount ofdietary intake, ophthalmological examination, acoumetric examination,hematological examination and hematochemical examination.

The autopsies of the above white rats revealed that there were noabnormal findings in the weight of each organ except hypertrophy ofspleen, however, there were also observed moderate level of local lungseptum and slight hemostasis in spleen, liver, kidney, andesophagogastric mucosal edema and extravasation of inflammatory cells.However, these lesions were also observed in an experimental grouptreated with a low dosage of composite medication of less than 5 μg/kgof calcitriol and less than 50 mg/kg of alendronate as well as in acontrol group and thus these lesions were not considered due to themedication. A slight esophagogastric inflammation observed appears not apeculiar change but is considered as a reversible injury in rodents inevaluating the toxicity of a given medication. There were no abnormalfindings in brain, bone marrow, sexual gland and peptic gland.

As mentioned above, the single oral administration of a composite drugof calcitriol and alendronate with a mixing ratio of 1:10,000 showedthat the LD₅₀ was shown to be more than 75 μg/kg and 750 mg/kg forcalcitriol and alendronate, respectively, thus proving that this is alow toxic drug. The repetitive oral administration for 5 weeks of acomposite drug containing 25 μg/kg of calcitriol and 250 mg/kg ofalendronate also did not incur any noticeable subacute toxicity atcompletion of convalescence as well as at completion of administration,and considering that the supposed human dosage (based on 60 kg ofaverage body weight of a person) is only equivalent to one 3000^(th) ofthat administered in white rats (i.e., 0.5 μg of calcitriol and 5 mg ofalendronate) the composite drug containing calcitriol and alendronateaccording to the present invention is thus thought to be safe.

Experimental Example 5 Tests of a Composite Drug Containing Calcitrioland Alendronate for the Various Mixing Ratios Between Calcitriol andAlendronate

1) The miscibility between a mixture of calcitriol and alendronate andadditives such as an excipient Excipients were added to the abovemixture of calcitriol and alendronate (5 μg: 50 mg) in a certain ratioas shown in the following tables 5 and 6.

2) To the resulting mixture in 1) was then added 5% of water content andstored it at 40° C. for 14 days.

The stability of the above mixture was examined by means of HPLC (highperformance liquid chromatography) and the results are shown in thefollowing tables 5 and 6.

TABLE 5 Stability of calcitriol (residual rate %) Classification MixingRatio Dry Water content (%) Calcitriol + — 80.1 79.6 AlendronateMannitol   9:1 101.9 101.6 White sugar   9:1 94.7 95.0 Cellactose   9:164.5 63.5 β-cyclodextrin   9:1 97.5 96.3 Povidone   9:1 98.3 99.1Hydroxypropyl 3.6:1 95.4 93.5 methyl cellulose Hydroxypropyl 3.6:1 91.892.2 cellulose Sodium lauryl 3.6:1 83.9 83.4 sulfate Low substituted3.6:1 95.4 94.3 Hydroxymethyl cellulose Croscarmellose 3.6:1 93.5 92.0sodium Calcium carboxy 3.6:1 75.2 73.5 methyl cellulose Crospovidone3.6:1 88.5 89.7 Butylated 3.6:1 27.7 14.4 hydroxytoluene Butylated 3.6:184.6 78.3 hydroxyanisole DL-α-tocopherol 3.6:1 86.4 86.2 Calciumstearate 3.6:1 101.7 101.1 Magnesium 3.6:1 100.3 98.9 stearate ColloidalSilicon 3.6:1 99.8 98.5 dioxide

TABLE 6 Stability of Alendronate (residual rate %) Classification MixingRatio Dry Water content (%) Calcitriol + — 99.0 100.2 AlendronateMannitol 1:1 96.2 99.3 White sugar 1:1 100.8 97.8 Cellactose 1:1 95.497.3 β-cyclodextrin 1:1 100.2 98.4 Povidone 1:1 97.7 99.9 Hydroxypropyl1:1 98.2 96.1 methyl cellulose Hydroxypropyl 1:1 75.7 85.2 celluloseSodium lauryl sulfate 5:2 100.4 99.7 Low substituted 5:2 98.1 98.7Hydroxymethyl cellulose Croscarmellose 5:2 100.0 97.4 sodium Calciumcarboxy 5:2 98.0 97.1 methyl cellulose Crospovidone 5:2 100.2 97.6Butylated 5:2 100.6 99.1 hydroxytoluene Butylated 5:2 97.9 97.2hydroxyanisole DL-α-tocopherol 5:2 94.2 99.0 Calcium stearate 5:2 95.498.3 Magnesium stearate 5:2 97.7 98.4 Colloidal Silicon 5:2 99.0 100.1dioxide

The results of the above tables 5 and 6 showed that mannitol as anexcipient, porvidone as a binder, calcium stearate or magnesium stearateas a lubricant are having an excellent property in its designated field,respectively. Butylated hydroxytoluene and butylated hydroxyanisole asantioxidants were thought to affect the stability of calcitriol atfirst, however, additional experiments confirmed that they only affectcalctriol at the level of its analysis and considering that they areused only trace amount it would not incur any problem. As a result,additives being incorporated into the pharmaceutical agent were selectedas follows: mannitol as an excipient, porvidone as a binder, calciumstearate as a lubricant, butylated hydroxytoluene and butylatedhydroxyanisole as antioxidants, and lauryl sodium sulfate as aresorption fortifier.

PREPARATIVE EXAMPLE

The pharmaceutical agents according to the present invention wereprepared in the form of naked tablets, enteric coated tablets, granules,enteric coated granules, capsules, enteric coated capsules.

Preparative Examples 1-3 Preparation of Naked Tablets

Naked tablets were prepared according to the composition and thecontents shown in the following table 7.

First, D-mannitol was added to a mixture consisting of calcitriol andethanol, and were then added ethanol, antioxidants and a binder.

Apart from the process of preparing calcitriol, other ingredients ofalendronate, mannitol, resorption fortifier were mixed, and then ethanoland a binder were added to it. Then, calcitriol and alendronate weremixed and a disintegrating agent and a lubricant were added and thustableted.

TABLE 7 Preparation Examples (mg) Classification 1 2 3 Main Alendronate6.53 6.53 6.53 Ingredients Calcitriol 0.0005 0.0005 0.0005 ExcipientD-mannitol 103.23 102.78 37.23 Antioxidant Butylated 0.12 0.12 0.12hydroxytoluene Butylated 0.12 0.12 0.12 hydroxyanisole Binder Povidone 33 3 Solvent Ethanol adequate adequate adequate Sustained- Hydroxypropyl— — 24 releasing methyl agent cellulose Methyl — — 48 celluloseDisintegrating Croscarmellose 6 6 — agent sodium Resorption Sodiumlauryl — 0.45 — fortifier sulfate Lubricant Calcium 1 1 1 stearate

Preparative Examples 4-6 Preparation of Enteric Coated Tablets

Enteric coated tablets were prepared the same as in the preparation ofthe above preparation examples 1-3 with the exception that the nakedtablets prepared in the preparation examples 1-3 were coated withenteric coating solution to have the ingredients and the contents asshown in the following table 8.

TABLE 8 Preparation Examples (mg) Classification 4 5 6 Main Alendronate6.53 6.53 6.53 Ingredients Calcitriol 0.0005 0.0005 0.0005 ExcipientD-mannitol 103.23 102.78 37.23 Antioxidant Butylated 0.12 0.12 0.12hydroxytoluene Butylated 0.12 0.12 0.12 hydroxyanisole Binder Povidone 33 3 Solvent Ethanol adequate adequate adequate Sustained- Hydroxypropyl— — 24 releasing methyl agent cellulose Methyl — — 48 celluloseDisintegrating Croscarmellose 6 6 — agent sodium Resorption Sodiumlauryl — 0.45 — fortifier sulfate Lubricant Calcium 1 1 1 stearateEnteric coating Hydroxypropyl 9.5 9.5 9.5 agent methyl cellulosephthalate Platicizer Triethyl citrate 0.5 0.5 0.5

Preparative Examples 7-9 Preparation of Granules

Granules were prepared as shown in the following table 9.

First, D-mannitol was added to a mixture consisting of calcitriol andethanol, and was then added with ethanol, antioxidants and a binder. Theentire mixture was then pulverized by using a conventional pulverizerinto a size of 1435 mesh and then dried at 40° C. to obtain calcitriolgranules.

Apart from the process of preparing calcitriol, other ingredients ofalendronate, mannitol, resorption fortifier were mixed, ethanol and abinder were again added to the above mixture, and then pulverized byusing a conventional pulverizer into a size of 1435 mesh and dried at40° C. to obtain alendronate granules. Then, calcitriol granules andalendronate granules were mixed, and a disintegrating agent and alubricant were added and thus tableted.

TABLE 9 Preparation Examples (mg) Classification 7 8 9 Main Alendronate6.53 6.53 6.53 Ingredients Calcitriol 0.0005 0.0005 0.0005 ExcipientD-mannitol 961.47 961.02 361.47 Antioxidant Butylated 1 1 1hydroxytoluene Butylated 1 1 1 hydroxyanisole Binder Povidone 25 25 25Solvent Ethanol adequate adequate adequate Sustained- Hydroxypropyl — —200 releasing methyl agent cellulose Methyl — — 400 cellulose ResorptionSodium lauryl — 0.45 — fortifier sulfate Lubricant Calcium 5 5 5stearate

Preparative Examples 10-12 Preparation of Enteric Coated Granules

Enteric coated granules were prepared the same as in the abovepreparation examples 7-9 with the exception that the granules preparedin the above preparation examples 7-9 were coated with enteric coatingsolution to have the ingredients and the contents as shown in thefollowing table 10.

TABLE 10 Preparation Examples (mg) Classification 10 11 12 MainAlendronate 6.53 6.53 6.53 Ingredients Calcitriol 0.0005 0.0005 0.0005Excipient D-mannitol 751.87 751.42 271.87 Antioxidant Butylated 0.8 0.80.8 hydroxytoluene Butylated 0.8 0.8 0.8 hydroxyanisole Binder Povidone40 40 40 Solvent Ethanol adequate adequate adequate Sustained-Hydroxypropyl — — 160 releasing methyl agent cellulose Methyl — — 320cellulose Resorption Sodium lauryl — 0.45 — fortifier sulfate Entericcoating Hydroxypropyl 146.4 146.4 146.4 agent methyl cellulose phthalatePlaticizer Fatty acid 24.4 24.4 24.4 glyceride Lubricant Talc 29.2 29.229.2

Preparative Examples 13-15 Preparation of Capsules

Capsules were prepared as shown in the following table 11.

First, D-mannitol was added to a mixture consisting of calcitriol andethanol, and were then added with ethanol, antioxidants and a binder.The entire mixture was then pulverized by using a conventionalpulverizer into a size of 1435 mesh and then dried at 40° C. to obtaincalcitriol granules.

Apart from the process of preparing calcitriol, other ingredients ofalendronate, mannitol, resorption fortifier were mixed, ethanol and abinder were again added to the above mixture and then pulverized byusing a conventional pulverizer into a size of 14-35 mesh and dried at40° C. to obtain alendronate granules. Then, calcitriol granules andalendronate granules were mixed, and a disintegrating agent and alubricant were added and finally filled into capsules.

TABLE 11 Preparation Examples (mg) Classification 13 14 15 MainAlendronate 6.53 6.53 6.53 Ingredients Calcitriol 0.0005 0.0005 0.0005Excipient D-mannitol 182.07 181.62 67.07 Antioxidant Butylated 0.2 0.20.2 hydroxytoluene Butylated 0.2 0.2 0.2 hydroxyanisole Binder Povidone5 5 5 Solvent Ethanol adequate adequate adequate Sustained-Hydroxypropyl — — 40 releasing methyl agent cellulose Methyl — — 80cellulose Disintegrating Croscarmellose 5 5 — agent sodium ResorptionSodium lauryl — 0.45 — fortifier sulfate Lubricant Calcium 1 1 1stearate Capsule Empty capsule 64 64 64 #2¹⁾ ¹⁾Product of SEOHEUNGCapsule Co., Ltd. (Korea)

Preparative Examples 16-18 Preparation of Enteric Coated Capsules

Enteric coated granules were prepared the same as in the abovepreparation examples 13-15 with the exception that the ingredients werecoated with enteric coating solution and then finally filled intocapsules with the ingredients and the contents as shown in the followingtable 12.

TABLE 12 Preparation Examples (mg) Classification 16 17 18 MainAlendronate 6.53 6.53 6.53 Ingredients Calcitriol 0.0005 0.0005 0.0005Excipient D-mannitol 178.07 177.62 63.07 Antioxidant Butylated 0.2 0.20.2 hydroxytoluene Butylated 0.2 0.2 0.2 hydroxyanisole Binder Povidone10 10 10 Solvent Ethanol adequate adequate adequate Sustained-Hydroxypropyl — — 40 releasing methyl agent cellulose Methyl — — 80cellulose Disintegrating Croscarmellose 5 5 — agent sodium ResorptionSodium lauryl — 0.45 — fortifier sulfate Enteric coating Hydroxypropyl36.6 36.6 36.6 agent methyl cellulose phthalate Platicizer Fatty acid6.1 6.1 6.1 glyceride Lubricant Talc 7.3 7.3 7.3 Capsule Empty capsule77 77 77 #1²⁾ ²⁾Product of SEOHEUNG Capsule Co., Ltd. (Korea)

Experimental Example 6 Stability Test of Enteric Coated Tablets

Enteric coated tablets prepared according to the preparation example 5were stored for two months under an accelerated condition (40° C.,relative humidity 75%) and were examined for the stability of tabletsthree times and the result is in the following table 13.

In evaluating the stability of tablets, the property was evaluated byobserving by naked eyes and was confirmed by means of HPLC. The contentsof both calcitriol and alendronate were also measured by HPLC.

TABLE 13 First Trial Second Trial Third Trial After After two two Aftertwo Test Items Initial month Initial month Initial month DescriptionWhite, White, White, White, White, White, round- round- round- round-round- round- shaped shaped shaped shaped shaped shaped film film filmfilm film film coated coated coated coated coated coated tablet tablettablet tablet tablet tablet Identification positive positive positivepositive positive positive Calcitriol 109.6 109.5 110.8 109.1 109.8109.8 Content (%) Alendronate 99.8 98.9 100.3 99.3 100.0 99.8 Content(%)

The results shown in the above table 13 revealed that the tablets werestable with the stability test for 2 months both at room temperature andunder an accelerated condition.

What is claimed is:
 1. A pharmaceutical composition for treatment ofmetabolic bone disease, which comprises a mixture of first granules andsecond granules wherein said first granules comprise an effective amountof calcitriol, as an active ingredient, and at least one excipient, andsaid second granules comprise an effective amount of alendronate, as anactive ingredient, and at least one excipient; and wherein saidcomposition contains about 1,000-50,000 parts by weight of alendronateper one part by weight of calcitriol; and further wherein said excipientin both granules comprises mannitol, said mannitol comprising about 70to 98 weight % of the total composition, and wherein about 50-60 weightpercent of the total mannitol is combined with said calcitriol, andabout 40-60 weight percent of the total mannitol is combined with saidalendronate.
 2. The pharmaceutical composition of claim 1, wherein saidcomposition is in a form of uncoated tablets, enteric coated tablets,enteric coated granules, capsules or enteric coated capsules.
 3. Thepharmaceutical composition of claim 1, further comprising, as aresorption fortifier, an additive comprising about 0.01 to 10 weightpercent of sodium lauryl sulfate based on the weight of the totalcomposition.
 4. The pharmaceutical composition of claim 1, wherein atleast one of said granules further comprises ethanol.
 5. Thepharmaceutical composition of claim 1, which is enteric coated granules.6. The pharmaceutical composition of claim 1, wherein said mannitol isD-mannitol.
 7. The pharmaceutical composition of claim 1, which furthercalcium stearate in an amount of 0.1 to 20% wt. % based on the totalcomposition.
 8. The pharmaceutical composition of claim 1, which furthercomprises magnesium stearate in an amount of 0.1 to 20 wt. % based onthe total composition.
 9. The pharmaceutical composition of claim 1,wherein said mannitol and said calcitriol and alendronate, have a mixingratio of mannitol: (calcitriol+alendronate) of 9:1.
 10. Thepharmaceutical composition of claim 1, which further comprises asustained-release additive selected from the group consisting ofhydroxypropyl methyl cellulose, methyl cellulose, hydroxypropylcellulose and ethyl cellulose.
 11. The pharmaceutical composition ofclaim 1, which further comprises butylated hydroxytoluene or butylatedhydroxyanisole.
 12. The pharmaceutical composition of claim 1, whichfurther comprises povidone, calcium stearate, butylated hydroxytoluene,butylated hydroxyanisole and sodium lauryl sulfate.
 13. A method oftreating metabolic bone disease which comprises administering aneffective amount of the pharmaceutical composition of claim 1 to amammal in need thereof.
 14. The method of claim 13, wherein saidmetabolic bone disease comprises osteoporosis, Paget's Disease,rachitis, osteomalacia, renal osteodystrophy, hyperparathyroidism andhypoparathyroidism.
 15. The method of claim 13, wherein said mammal is ahuman.
 16. The method of claim 13, wherein said metabolic disease isosteoporosis.